Pulsed field gel electrophoresis (PFGE) dates from 1984, when Schwartz D. C. and Cantor C. (Cell, 37, 67-75, 1984; U.S. Pat. No. 4,473,452) observed that applying electric pulses that periodically alternated their direction in a certain angle in relation to an agarose gel, large intact DNA molecules were resolved as band patterns. The authors also determined that the separations of the molecules essentially depended on the duration of the electric pulses. Later, it was determined that the geometry of the lines of force of the approximates alternating electrical fields, the strength of them, the experimental temperature, the ionic strength of the buffer solution and the concentration of the agarose gel were important factors that influenced the resolution that could be achieved among DNA molecules. (Birren B. and Lai E. Academic Press. New York, 1993, p 107, 111, 129, 131, 135; López-Cánovas L. et al., J. of Chromatogr. A, 1998, 806, 123-139; López-Cánovas L. et al., J. of Chromatogr. A, 1998, 806, 187-197).
Pulsed field electrophoresis renders the separation of the DNA molecules as band patterns. Each pattern is formed in the lanes of the separation gels after the electrophoresis. Altogether, the agarose plugs containing the immobilized DNA molecules are loaded into each well of the gel, then, the molecules migrate along the length of the gel and form the band patterns during the electrophoresis. That is why this type of electrophoresis has associated a method for the preparation of intact DNA molecules immobilized in plugs of gel. These molecules can be or not, digested with restriction endonucleases before the electrophoresis.
Several systems to perform PFGE have been developed. They are characterized for having chambers in which the electrodes are placed in different arrangements. Among these chambers are the OFAGE (Orthogonal Field Alternating Gel Electrophoresis, Carle C. F. and Olson M. V. Nucleic Acids Res. 1984, 12, 5647-5664) CHEF (‘Contour Clamped Homogeneous Electric Field’, Chu G. Science 234, 1986, 1582-1585), TAFE (‘Transversal Alternating Field Electrophoresis’, U.S. Pat. No. 4,740,283), FIGE (‘Field Inversion Gel Electrophoresis’, U.S. Pat. No. 4,737,251 of Carle G. F. and Olson M. V) arrangement of electrodes, and the minichambers MiniTAFE and MiniCHEF (Riverón, A. M. et al., Anal. Left, 1995, 28, 1973-1991; European Patent Application EP 0 745 844).
All these systems are characterized by having electronic circuitry for alternating the electric fields and accessories for the preparation of the gel. There are also accessories for the preparation of the samples. The systems differ among them by the complexity of the electronics to energize the electrodes and to switch the orientation of the electric field. They also differ by their capacity to render straight paths of migration of the band patterns. The possibility to obtain straight paths of migration is essential when the comparison of the patterns of several samples is wished, while the simplicity of the electronics facilitates and makes cheaper the construction of the systems. Among the systems mentioned, only three render straight paths of migration of the molecules:
1.—the CHEF system, which has electrodes that are clamped to predetermined electrical potentials, electrodes that are arranged in a hexagonal contour around a submarine gel that is horizontally positioned;
2.—the TAFE system, in which the electrophoresis is performed in submarine gels that are positioned vertically in the chamber and uses fields transversal to the surfaces of the gel; and
3.—the FIGE system, in which the electrophoresis is performed in horizontal submarine gels that are positioned in conventional electrophoresis chambers, which have two electrodes. In this system, the orientation of the electric field is reverted periodically.
These systems have in common that in their chambers the gel is symmetrically crossed by the lines of force of the electric fields that are generated at the electrodes with opposite polarity in the electrode arrangement. In that gel, the samples containing the intact DNA molecules are loaded. In all these chambers exist zones where the force lines of the electric field do not act on the molecules. The zone of the chamber that contains the gel and is crossed by the lines of force of the electric field that directly interact with the molecules will be denominated here as useful electrophoresis zone (UEZ). Whereas the zones of the chamber crossed by the lines of force of the electric field that do no act on the molecules will be denominated here as non-useful electrophoresis zones (NEZ). All existing chambers to perform PFGE have a single UEZ and several NEZ regions.
The chamber and the electronics of the FIGE system are simple. FIGE chambers that allow the simultaneous analysis of many samples exist (up to 96 samples, using two combs of 48 teeth in the chamber OnePhorAll Submarine Gel System of Jordan Scientific, BDH Catalogue BDH, 1997, Section E p 4-371), but in these chambers inversion of the mobility of the molecules occur (Carle G. F., Frank M. and Olson M. V. Science, vol. 232, p 65-68, 1986). Due to the absence of a theory that predicts the inversion mobility in FIGE under any experimental conditions, such inversion limits the use of these chambers to analyze the size of DNA molecules separated and to compare their band patterns. For instance, this phenomenon will cause that two DNA molecules of different sizes migrate the same distance in the gel, preventing their identification, excepting by means of hybridization with DNA probes. Up to now, the two only ways to estimate the size of large DNA molecules separated in experiments of PFGE are:    1) To compare the distances migrated by the molecules under study to the distances migrated by the size markers and    2) To use equations that describes the distances migrated by the molecules under different electrophoresis conditions and later replace in the equations the migrated distances and the experimental variables.
In FIGE the size markers can also suffer mobility inversion and, as mentioned above, there is no theory capable to predict the moment and conditions of appearance of such inversion. These are serious drawbacks of FIGE chambers, especially to compare many samples, for instance, in molecular epidemiology studies. Because of these reasons, the systems most frequently used to compare band patterns of many samples are CHEF and TAFE.
Gardiner K. et al. in their paper published in Somatic Cell Mol. Genet. 1986, 12, 185-195 proposed the TAFE system. They called it “Vertical Pulsed Field Electrophoresis” (VPFE) and developed an equipment which was disclosed in U.S. Pat. No. 4,740,283 dated Apr. 26, 1988. This system for the separation of DNA molecules uses a vertical gel of 10×7.6×0.6 cm (height×width×depth) and has the electrodes arranged in parallel to the faces of the gel and across the chamber. In the chamber, each member of a pair of electrodes with opposite polarity is positioned in front of a face of the gel. The cathode is positioned at the top and near the origin of migration and the anode far from it, at the bottom. Such electrode arrangement generates equipotential lines spanning the length of the gel and a gradient potential or electric field, where the lines of force of such electric field cross the gel transversally. Then, along the height of the gel a gradient of electric field strength and of the angle formed between the lines of force of the two pair of electrodes are obtained. That is the reason why the molecules are compelled to migrate during each pulse through the thickness of the gel. The resultant migration occurs in vertical direction, downward. Despite of the existence of these gradients, all the gel points situated widthwise and at the same height, in relation to the plane that contains both cathodes or both anodes, are at a same value of electric potential (equipotential lines). Thus, molecules of the same size migrate similar distances during the electrophoresis in all the lanes of the gel and migrate following straight paths up to the same height in the gel, independently on the wells in which the samples were loaded.
Based on these principles, Beckman Instrument, Inc. (Beckman, The Geneline System Instruction Manual, ed. Spinco Division of Beckman Instruments, 1988), constructed the equipment called “Geneline I”, or “Transverse Alternating Field Electrophoresis System” known as TAFE. This system uses a gel of 11×7.2×0.6 cm (height×width×thickness), which is placed between the pairs with opposite electrodes that are separated 20 cm. Later, Beckman Instruments, Inc. developed the equipment “Geneline II” in which the gel was enlarged to 14.2×15×0.3 cm. The Geneline II equipment is no longer been produced.
To resolve large DNA molecules in a band pattern, a long time is required in the TAFE equipments Geneline I and Geneline II. For instance, Geneline I needs 24 hours to render a pattern of 11 bands corresponding to the chromosomes of the yeast Saccharomyces cerevisiae (molecules less than 1.6 Mb in length. 1 Mb=106 base pairs). This equipment may need up to 90 hours to separate the DNA molecules of the amoebic genome in 17 bands (Orozco E. et al., Molec. Biochem. Parasitol. vol. 59, p 29-40, 1993). TAFE chambers require a large volume buffer solution to cover the electrodes (approximately 3500 ml in Geneline II) and through the electrophoresis buffer the flow of electric current is high and the generated heat might be large. If in the TAFE equipment, the potential difference applied across the electrodes with opposite polarity is increased, the maximum current output of the power supply may be achieved. That is why, the companies recommend 10 V/cm as the maximum value of electric field (for power supplies with a maximum current output of 0.4 Amp). Large heat generation in the electrophoresis impedes the reduction of the duration of electrophoresis by increasing the electric field strength. It has been stated that the use of elevated voltages or high temperatures broaden and make diffuse the bands of the electrophoresis pattern, rendering poorly resolved bands. The advantage of the Geneline II is to permit the simultaneous analysis of 40 samples, which facilitates the comparative analysis of the electrophoresis patterns given by many samples.
Gilbert Chu (Science 1986, 234, 16, 1582-1585) developed CHEF system on the following basis: a homogeneous electric field is theoretically generated by two infinite electrodes placed in parallel at certain distance. To simulate a homogeneous field using finite electrodes, another group of electrodes is placed in a plane, along a closed polygon, that might be a square or a hexagon.
The x axis (y=0) of the plane is set to coincide with a side of the polygon and zero volt is applied. The opposite side is placed at a distance ‘A’ (y=A) from the origin of ordinates and it is polarized to a potential ‘Vo’. The rest of electrodes are polarized according to V(y)=Vo·y/A. In this way, the potential generated in the interior of the polygon is equal to those that should be generated by two infinite parallel electrodes separated a distance ‘A’. The reorientation angle obtained by electronic permutation of the polarity between two pairs of different sides will be 90° for the square and 60° or 120° for the hexagon. A method to clamp the desired potentials across the CHEF electrodes is to set a series of resistors wired to form a voltage divisor between potentials V(0)=0 and V(A)=Vo. From each of the nodes, formed by the union of two resistors, the voltage for the polarization of one electrode is withdrawn.
Based in these principles, the Bio-Rad Company developed the equipments CHEF-DR II, CHEF-DR III and CHEF Mapper (U.S. Pat. No. 4,878,008, U.S. Pat. No. 5,084,157 and U.S. Pat. No. 5,549,796). The last is the most advanced system. To clamp the voltages across the electrodes of the hexagonal array, the voltage divisor is wired to a transistorized system and operational amplifiers. This electronic design warrants that the voltages that are applied across the electrodes of the hexagonal array will be always correct.
The dimensions of the CHEF Mapper electrophoresis chamber are 11.4×44.2×50.3 cm (height×width×depth), it weights 10.2 Kg and uses 2.2 liters of buffer solution. This system uses a gel of 14×13 cm (width and length) that is concentrically positioned with the hexagonal arrangement of 24 electrodes, whose parallel sides are separated 30 cm or more. CHEF Mapper is also capable to use a wider gel where up to 40 samples can be loaded into.
The TAFE and CHEF equipments are able to separate chromosomal sized DNA molecules. Nevertheless, a common disadvantage of the CHEF and TAFE equipments is that the chambers are unnecessarily large, because their dimensions have not been optimized yet, particularly when thin sample plugs are used. It has been demonstrated that the thickness of the agarose plugs that contain the DNA samples influences the resolution of the bands, the electrophoresis time and the length of the gel to be used (López-Cánovas L. et al. J Chromatogr. A, 1998, 806, 187-197). In that work, it was demonstrated that if it is wished to obtain a resolution ‘X’ between two any molecules, this value is obtained in less space and less time if the bands are thinner, which is achieved if the plugs are also thinner. Among the consequences of using large electrophoresis chambers are:    I) When high electric fields are applied, the use of power supplies with large maximum output is required. These chambers have more than 20 cm of distance between the electrodes with opposite polarity; therefore, the maximal electric field that can be applied in these equipments is approximately 10 V/cm.    II) The experiments are long in these chambers. Two factors influence long run duration: very low electric fields are used (usually 6 V/cm), and samples are around 0.1 cm thickness. For instance, normal experiments take 24 hours to obtain the electrophoresis patterns of the eleven chromosomal bands, corresponding to DNA molecules of Saccharomyces cerevisiae less than 1.6 megabases (106 base pairs), and up to 90 hours to separate the 17 bands of DNA molecules from the genome of Entamoeba histolytica (Orozco E et al, Mol. Biochem. Parasitol. 1993, 59, 29-40).    III) The equipments are not economical, because large amount of expensive reagents (such as Tris and agarose) and biological samples are used. The latter might impede certain applications (for instance in clinical diagnosis).    IV) A large quantity of heat is generated in the electrophoresis chamber when the driving force of the electrophoresis or electric field (which depends on the applied voltage across the electrodes and on the current intensity that flows through the buffer solution) is increased. If the electric field is increased (aimed to increasing the velocity of separation), it should be done by increasing the voltage applied across the electrodes, and therefore the current intensity. By Joule effect, the generation of heat in the chamber will increase. An excessive increase of heat evolved will broaden and make the bands diffuse and will provoke distortion of the electrophoresis pattern and even entrapment of DNA molecules in the pores of the gel and the complete absence of migration.
Nevertheless, the large volume of buffer solution filling these chambers has the advantage that the turbulences generated in the solution during its circulation are attenuated. Altogether, the gel is so distant from the electrodes that any local change in conductivity near the electrodes produced by electrolysis is diluted and will not have deleterious effect due to the large volume of solution.
In 1995 were disclosed the MiniCHEF and MiniTAFE equipments, in which Pulsed Field Gel Electrophoresis of 8 samples loaded into a gel are performed (Riverón A. M. et al., Anal. Lett, 1995, 28, 1973-1991; European Patent Application EP 0 745 844). These equipments overcame the deficiencies of the above-mentioned systems. The MiniCHEF as well as the MiniTAFE use thin samples thinner than 0.1 cm and they allow the application of higher electric fields rendering adequate resolutions among the bands of the patterns. Therefore, in the miniequipments, the chromosomes of the yeast Saccharomyces cerevisiae were resolved in 4 to 5 hours.
The separation between the opposite electrodes of minichambers is small, thus allowing the construction of smaller chambers and the use of less volume of buffer to cover the electrodes and the gel (Riverón A. M. et al., Anal. Lett, 1995, 28, 1973-1991; European Patent Application EP 0 745 844, Bull. 1996/49). That is why in MiniCHEF and MiniTAFE, low amount of heat is not evolved, even if high electric fields are applied. The samples loaded into the gels of these equipments need a small amount biological material (Riverón A. M. et al., Anal. Lett, 1995, 28, 1973-1991). Furthermore, they save laboratory bench space.
The authors of these equipments demonstrated the feasibility of performing PFGE in gels that are not long. For instance, gels of 4 cm in length were used. By means of the use of mini-equipments López-Cánovas L. et al. (López-Cánovas L. et al., J Chromatogr A, 1998, 806, 187-197) demonstrated that plugs thicker than 0.1 cm render thick bands and the molecules need more time and more gel length to be separated. In addition, the use of thick samples does not improve the quality of the electrophoresis pattern and does not reveal more bands.
The mini-equipments proposed by Riverón A. M. et al. to perform Pulsed Field Gel Electrophoresis have chambers whose sizes are calculated based on the existence of other equipments of larger dimensions (Riverón A. M. et al., Anal. Lett, 1995, 28, 1973-1991; European Patent Application EP 0 745 844). Therefore, they can inherit errors of the equipments from which they were designed. In fact, the mini-equipments inherited from the large chambers an open system for the preparation of the gel and the absence of a proper system for attenuating turbulences of the buffer flowing throughout the chamber. In the patent application and the related papers, the effects of the reduction of the volumes of the buffer and the gel on the electrophoresis pattern are not mentioned. That is, the question whether this volume of buffer is enough to attenuate the turbulences during its circulation, or whether the irregularities in the gel and the differences in dimensions of the plugs influence the quality of the bands patterns that are obtained, are not resolved yet.
These troubles increase with miniaturization, because miniaturization magnifies the manufacture errors. For instance, if a meniscus of 0.1 cm height is formed in a gel of 1 cm of thickness, the error in the height of the gel would be 10%, while that same error in a 0.4 cm thick gel represents 25%. Therefore, the magnification of the errors by miniaturizing the system can become critical factors to obtain reproducible bands patterns.
The relevant parameter of pulsed field electrophoresis equipments is the separation between the electrodes, because it determines the values of electric field that can be applied. It also determines the driving force of the molecules, the dimensions of the chambers, the systems that should be used to homogenize the variables of the electrophoresis, the length of the separation gel, the thickness of the plugs where the samples are included and the width of each sample.
If the separation between the electrodes with opposite polarity is not optimal, for instance, if it is too large, then the dimensions of the gel, the chamber and the number of samples that can be applied in those gels will not be optimal. If the plugs do not have the proper thickness and size, an excessive quantity of gel will be used and large electrophoresis time will be consumed. In addition, the shape and distribution of the dimensions of the chambers as well as the existence of a single UEZ region determines that the reagents consumed in these chambers will not be used optimally. Therefore, the desired goal is to develop chambers with optimal dimensions, which allow the application of high electric fields; chambers whose internal dimensions vary according to the number of samples they analyze and that the electrophoresis run time to be short without loosing resolution or high capacity of sample analysis.
From the above reasons, it is concluded that:                Large chambers of the current PFGE systems are not optimal, because the separation between electrodes with opposite polarity is unnecessary large and the same amount of reagents is used, independently on the number of samples to be studied.        The chamber dimensions are not optimal. The dimensions of the chambers (height, width and depth) do not warrant that the current flowing through the chamber does not exceed easily the output limits of the power supplies for PFGE, and thus, and do not separate the molecules fast at high electric fields.        In order to increase the number of UEZ, relevant constructive modifications have to be carried out in the existing chambers. They may affect the proper functioning of the systems. This factor influences the optimization of the use of reagents.        
As it was already mentioned, the TAFE chambers (Geneline I, Geneline II) and MiniTAFE have an electrode platform to accommodate a gel (or two gels in Geneline II). Electrode platform whose width is equal to the width of the chamber and its height depends on the separation between the electrodes with opposite polarity (that is, they have an UEZ region). In the gel(s), so samples can be applied as many as is allowed by its width, the width of the samples and the separation between them. The equipments that have an UEZ region use a constant volume of buffer solution to cover its electrodes.
If the number of samples that is desired to be simultaneously analyzed exceed the maximal capacity of analysis of the UEZ of any of the mentioned chambers (for instance, more than 8 in MiniTAFE, more than 20 in Geneline I and more than 40 in Geneline II), it would be necessary to perform several electrophoresis. Therefore, the comparison of the resulting band patterns will not be reliable. For instance, when it is desired to characterize the genome of 100 isolates of a particular microorganism, either from a collection of isolates of the biotechnological industry, or infected human, animal or vegetables. Then, these three chambers have insufficiencies in their capacity to simultaneously analyze more than 8, 20 or 40 samples, respectively, or are insufficient the possibilities to increase the capacity of analysis. Therefore, when it is necessary to perform co-electrophoresis of many samples to compare the band patterns of the DNA molecules of the samples, the maximal capacity analysis of TAFE (Geneline I, Geneline II) and MiniTAFE can be exceeded.
A known solution, that would increase twofold the capacity of sample analysis of the mentioned chambers, is the implemented in the FIGE chamber OnePhorAll. This consists in positioning two combs in the gel of the UEZ, one of them at the beginning of the gel and the other in the middle of it. However, in the TAFE system, the samples loaded into the wells formed by the two combs would not be subjected to the same electric field nor to identical reorientation angle; thus molecules of similar size would migrate different distances in the gel and the bands patterns would not be comparable.
Another possible solution could be to construct wider chambers with wider gels and UEZ zones. This solution was implemented in Geneline II and supposedly; it should allow the analysis of many samples (more than 40). That is why Geneline II was designed as a non-deep but wide and tall chamber. However, it was necessary to place dielectrics between the electrodes and the gel in order to obtain the characteristic angle gradient of TAFE system. These dielectrics considerable slow down the electrophoresis runs. On the other side, the electric current flowing through the chamber depends directly on the cross section area that encounters the ionic flux. Thus, through these very tall and wide chambers flow elevated current, exceeding those flowing in Geneline I and the former VPFE. Therefore, by applying low voltages, the maximum current (Imax), or power (Pmax) outputs of the power supply, are reached in less time. For instance, Macrodrive I, LKB: Imax=0.4 Amp, Vmax=500 volts, Pmax=200 Watts; PowerPack 3000, Bio-Rad, Cat. 1998-1999: Imax=0.4 Amp, Vmax=3000 volts, Pmax=400 Watts; Consort E802, Cat. BDH 1997: Imax=2 Amp, Vmax=300 volts, Pmax=300 Watts (Vmax, Imax and Pmax: maximum voltage, current and power outputs, respectively). Then, in this type of chambers is impossible to increase the electric field strength to reduce the electrophoresis time. Low electric fields unnecessary enlarge the duration of PFGE experiments, fact that reduces the spectrum of applications of these chambers in the fields of the science and technology that require the rapid obtainment of results. In addition, Geneline II uses larger volume of reagents than current chambers. In fact, Beckman Instruments has discontinued Geneline II.
Wider miniTAFE chambers could be designed (maximum applicable electric field: 25 V/cm for approximately 6 cm in width), because the chambers are neither deep nor tall. The cross section area of miniTAFE could be increased if the electric current (I) flowing through the buffer at a given ‘E’ value (for instance 8-10 V/cm) does not exceed the maximum output of the existing power supplies. These chambers use less volume of buffer solution than the current TAFE, Geneline I and Geneline II chambers. In miniTAFE, the band patterns would be obtained in a relative short time. However, such broad UEZ would need a very wide minigel, which would present difficulties in its casting and handling. Additionally, several minigels could be accommodated, but according to formula I (see forward), this chamber would not be efficient when analyzing a small number of samples. When it is necessary to analyze a small number of samples, for instance 8, the analyzing capacity of the gels of the TAFE Geneline I and Geneline II is largely wasted, because they have a single UEZ that can accommodate 20 or 40 samples, respectively. The reagents used in PFGE experiments are expensive. The equipments would efficiently use their capacities of separation of DNA molecules if the volumes of reagents used each time in the chambers would depend on the number of samples to be analyzed. This is impossible in chambers of a single UEZ because they use a constant volume of reagents. The volume of reagents in excess (ER %) that is used in chambers of a single UEZ can be defined asER(%)=100.0·(Nt−N)/Nt  (I)where:                Nt: Maximal number of samples that can be loaded in a minigel.        N: Number of samples really analyzed in an experiment        (Nt−N): Number of samples not loaded in the gel        
The ER values of the Geneline II and MiniTAFE systems are shown in Table 1. When few samples are analyzed, ER increases in both chambers, evidencing that they use reagents in excess when few samples are applied. Although miniTAFE (data in column 2, Table 1) uses less volume of reagents than TAFE, this volume neither varies with the number of samples analyzed. Hence, the volume of reagents used by the TAFE Geneline I, Geneline II and MiniTAFE chambers is constant and independent on the number of samples to be analyzed, fact that impedes its optimal use.
In addition, the buffer solution becomes exhausted during electrophoresis. That is why, to make an optimal design of the shape and dimensions of the chambers, it is necessary to know the time that the buffer solution takes to exhaust.
The chambers of the TAFE system to separate DNA molecules use a vertically placed gel and its cathodes are located at the top of the chamber. Then, the direction of migration is parallel to the vector of the gravity force. To avoid accidents with the electrodes while placing the gel in the chamber, Geneline I have two removable electrode platforms and the gel is accommodated into the chamber before such platforms are placed.
TABLE 1Excess of reagents (ER %) used in Geneline II and MiniTAFE.N (No. ofTAFE GL-IIMiniTAFEsamplesBc = 3500Bc = 325loaded)ER (%)ER (%)197.587.5295.075.0392.562.5490.050.0587.537.5685.525.0782.512.5880.00.0977.5—1075.0—1172.5—1270.0—1367.5—1465.0—2732.5—400.0——: Means that the gel does not have those wells.ER: Percentage of reagents used in excess.GL-II: Geneline II.Bc: Total volume of buffer solution filling the chamber, in ml.
To implement this solution it is necessary to place the electrodes and the gel in relation to the platforms in the proper position. However, this double positioning of the electrodes in the platforms and the platforms with respect to the gel can vary the relative disposition between the gel and the electrodes. Therefore, this aspect should be improved in the design of the chambers.
As it was mentioned, in the existing chambers there are zones crossed by lines of force of the electric field that do not act directly upon the molecules loaded into the gel (non useful electrophoresis zone, NEZ). These regions do not play an essential role in the separation of the DNA molecules.
The miniequipments previously reported are not optimum, because they do not have any system to attenuate turbulences of the buffer solution flowing through the chamber, neither to prepare gel without irregularities nor to form thin sample plugs of similar sizes and shapes.
Up to now, the attention has been focused on maintaining constant the voltage across the electrodes of the chambers during the electrophoresis. It is particularly notorious in the CHEF chambers, in which is necessary to set a given voltage value in each electrode of the hexagonal array. However, the quality of the band patterns and the experimental reproducibility are affected by variations in the voltage and other factors. The reproducibility of the band patterns is also affected by the factors that provoke non-homogeneity of the current flowing through the buffer filling the chambers and the factors that could distort the lines of force of the electric field.
These other factors have not been completely considered in PFGE systems. For this reason, the current systems can give as results distorted band patterns. These problems are relevant in the miniequipments for electrophoresis. They are:                The chambers do not have simple devices to attenuate the turbulences of the buffer flowing through the chamber and the external heat exchange.        The accessories to cast the gels do not avoid the formation of irregularities and defects in the electrophoresis gel.        The accessories to immobilize the DNA molecules in the agarose plugs do not warrant the formation of sample plugs with dimensions similar to those of the wells of the agarose gel. There are not devices to achieve a good alignment of the sample plugs in the migration origin either.        There are not devices to warrant maintaining the electrodes stretched.        
The mentioned aspects affect the obtainment of straight and reproducible band patterns in the different lanes of the gel. These aspects also affect the band pattern reproducibility during different electrophoresis runs in the same or in several equipments.
On the other hand, the chambers for pulsed field gel electrophoresis are filled with a buffer solution that is circulated between the chamber and an external heat exchanger. From the potential applied across the electrodes is generated the electric field or driving force of the molecules and the buffer solution is the medium through which the electric field is established. The physicochemical processes occurring in the buffer during the electrophoresis, as the electrolysis, the buffer heating by Joule-effect and the variations of the concentration of the ions of the buffer provoke non-homogeneities in the conducting properties of the buffer solution. The temperature, concentration and others variables affect the viscosity of the buffer, and thus the electric field generated through the buffer as well as the movement of the DNA molecules. Thus, DNA migration is affected in different fashions throughout the chamber, when any of these variables are randomly changed. The electrolysis also affects the buffer conductivity. The buffer in the chamber is constantly exchanged with thermostated buffer at constant temperature. It is accomplished by using a peristaltic pump. Therefore, it is intended to maintain homogeneous and constant the properties of the buffer solution. The buffer flow velocity should warrant the total exchange of the chamber's buffer in a few minutes. However, at a given buffer injection velocity, turbulences in the chambers are generated. Then, local non-homogeneity of the applied electric field is generated, which affects the movement of the DNA molecules.
The resulting band patterns depend on the variations of the conductivity in the buffer solution of the chamber and the presence of turbulences in that buffer. The turbulences are increased if the buffer is circulated at high flow velocities. The turbulence, vortices or waves locally change the height of the buffer, modifying randomly and regionally the electric resistance values of the buffer in the chamber. The variations in the electric current flowing through the different regions of the chamber modify the DNA migration and generate distorted DNA band patterns.
The equipment CHEF MAPPER from Bio-Rad considers this problem (CHEF Mapper XA Pulsed Field Electrophoresis System. Instruction Manual and Application Guide p 4. Bio-Rad). The Bio-Rad CHEF has two small chambers below the main chamber floor at the front and rear of the main chamber. They are used for buffer circulation and priming the pump. Buffer enters the main chamber through six holes in the floor near the top. A flow baffle just in front of these holes prevents gel movement. However, this system is not efficient to attenuate buffer turbulence, mainly when high flow velocity is used.
Neither the TAFE nor the miniequipments have any system for attenuating the turbulences of the buffer flowing throughout the chamber, which is a disadvantage. It is easy to sense that the turbulences are more harmful in the miniequipments that use less amount of buffer. For example, the turbulences in the CHEF Mapper chamber, filled with 2.2 L, are attenuated easier than in the miniCHEF and miniTAFE that use ten times less buffer.
As it was mentioned, the PFGE large chambers attenuate in certain extent the height of buffer oscillations. However, the miniequipments for PFGE are relatively recent, maybe for this reason the development of a system for attenuating the turbulences of the buffer flowing through these chambers has not been a focus of attention.
The gels used in the CHEF and TAFE equipments of large dimensions as well as in the mini-equipments matches with the mold where they are cast, a comb is inserted and the molten agarose is poured. While the agarose is solidifying, the mold is not covered. Then, because of the surface tension of the molten agarose, it wets the walls of the mold forming a meniscus. The meniscus is formed between the wells of the gel or in the walls of the gel mold. The mold to prepare the TAFE gel has a lid, but it has not accessories to avoid the meniscus formation among the teeth of the comb. The accessories to pour the gels of the miniCHEF and miniTAFE do not have cover, and then the meniscuses are formed in the sites above-mentioned.
The gel is the medium through which occurs the migration of DNA molecules during the electrophoresis. The presence of meniscuses at the edges of the gel or between the wells of the gel modifies the electric resistance in the gel and consequently the electric current. The regional changes of the electric current flowing through the gel affect the DNA migration in such regions. These changes are essential if meniscuses are formed. The gel wells are the origin of migration of the molecules; consequently, if the irregularities in these zones provoke changes in the velocity of migration of the molecules, the migration boundaries will be distorted. Then, these distortions will be maintained during the electrophoresis process, finally giving distorted patterns in the gel lanes. Any gel irregularity in any other region will also affect the molecule migration through such region. From the point of view of the band pattern reproducibility, the accessories to prepare the gel and the method to use it are important. The designs of efficient systems for pulsed field gel electrophoresis has been focused in afford chambers with different electrode configurations and an electronic circuitry suitable to switch the electric field and impose the voltages. The properties of the accessories to prepare the gels have not been exhaustively considered.
As it was mentioned, the pulsed field gel electrophoresis includes the methodology for the preparation of intact DNA molecules immobilized in gel plugs. To do it, it is necessary to have molds to form the sample plugs.
The existing molds are the following:                A mold to form similar and single plugs (Cantor C. R. and Schwartz D. C., U.S. Pat. No. 4,473,452);        A mold to forms long and flat ribbons that are cut to provide single plugs;        A mold to form long agarose rods that are cut to provide single plugs (Birren B. and Lai E. Pulsed Field Gel Electrophoresis: A Practical Guide, Academic Press, New York, 1993, 29-30).        
Usually, the above molds generate sample plugs of dimensions larger than the wells of the gel. For this reason, it is recommended to cut the plugs with the aid of a blade or other instrument (CHEF Mapper XA Pulsed Field Electrophoresis System. Instruction Manual and Application Guide p 40, Section 7. Catalog Numbers 170-3670 to 170-3673. Bio-Rad).
In the current chambers (CHEF, TAFE, miniCHEF and miniTAFE), the inequalities of the sample plugs provoked by cutting them after their preparation, affect the quality of the electrophoresis patterns. It is known that the sample plug thickness has influence upon the DNA resolution and the electrophoresis time. However, the effect of the shape and dimension inequalities of the sample plugs upon the electrophoresis patterns has not been thoroughly studied. The effects provoked by a bad alignment of the sample plugs lengthwise the origin of migration have not been studied yet. The researchers have used the sample plugs makers mentioned in the above paragraph. However, these molds do not include devices to cut the sample plugs with identical shape and dimensions and matching with the gel wells.
If it is considered that the band patterns obtained in each gel lane at the end of the electrophoresis depend on the fact that molecules of similar sizes are moving together from the wells toward the bottom of the gel, the importance of the accessories to prepare the sample plugs and align them in the gel wells will be understood. That is, the migration boundary should move forming a thin and straight band. When the migration boundary is deformed in the origin of migration, it will be maintained deformed during the electrophoresis, because in the chamber does not exist any device or force to correct the movement. The flaws preparing the sample plugs and troubles in their alignment in the gel wells are exactly reproduced in the bands separated in the patterns, and might provoke tilted and undulated bands.
In the U.S. Pat. No. 5,457,050 of 1995 of GH Mazurek, was disclosed a mold and a processing chamber to perform the cell immobilization and treat the cells inside the mold. Depending on the material used to construct such mold, it could be disposable or reusable. Besides that sample plug preparation could be longer using this processing chamber, said mold does not have associated a device to cut the sample plugs and, thus, plugs of similar sizes are not warranted to be obtained.
On other hand, the equipments TAFE Geneline I and Geneline II fix its four platinum electrodes between two parallel acrylic sheets (Beckman, The Geneline System Instruction Manual, ed. Spinco Division of Beckman Instruments, 1988). One of the end of each electrode goes toward the lid of the chamber, outside of the useful electrophoresis zone. It is done with the aim of connecting the platinum wire to a plug in the lid of the chamber. In this way, it is warranted the electrical continuity between the circuitry and buffer solution as well as the polarization of the electrodes. The platinum wire in the lid is insulated with a plastic capillary with high dielectric constant. As it is known, the platinum electrodes become thinner during pulsed field gel electrophoresis and during their use, the electrodes slacken and become bent and undulated for several zones. Them, the system used in the TAFE to set the electrodes has the disadvantage that to pull tight the electrode the experimenter must dismount the electrode from the lid and this is very difficult.
When the electrodes become bent, undulated or slacken, the equipotential lines in the gel and the force lines of the electric field become also distorted provoking that bands do not migrate in a sharp and straight boundary.
By the other side, the way to fix the electrodes in the TAFE equipments wastes a portion of platinum wire. For example, the Geneline I uses approximately one meter of platinum wire, while the active electrodes require only thirty centimeters. The Geneline II has a similar design.
In the CHEF Mapper, the electrodes (J-shape) are fixed on supports made of material with high dielectric constant, in such a way that one of its ends passes through the support (CHEF Mapper XA Pulsed Field Electrophoresis System. Instruction Manual and Application Guide p 4 and 65, Section 7. Catalog Numbers 170-3670 to 170-3673. Bio-Rad). The supports are inserted into the floor of the chamber. In this way, the platinum wire passes through the floor of the chamber and is connected to the circuitry to clamp the voltage across the electrodes. To seal the floor of the chamber a silicone sealant and rubber O-rings pressed down with a nut are used. The fixing of electrodes in the CHEF Bio-Rad saves platinum wire because the electrodes are not so large and they do not have to pass out of the buffer. However, it is not warranted that these electrodes are maintained stretched and consequently slight deformations of the lines of force of the electric field can occur.
The disclosed MiniTAFE and MiniCHEF equipments (Riverón A. M. et al., Anal. Lett, 1995, vol. 28, 1973-1991; European Patent Application EP 0 745 844, Bull. 1996/49) have extended the electrode platinum wire above the buffer solution level in the chamber as the TAFE equipment does. In this way, it is warranted the necessary communication between the electrodes and the electronic circuitry to polarize them. The regions of the platinum wire that do not act as electrode are insulated with tubing made of material with high dielectric constant to avoid the contact of such platinum wire with the buffer. The TAFE chamber uses electrodes that are at least as long as the width of the gel and are suspended between the sidewalls of the chambers. During the use, the electrodes slacken and undulated, so the band patterns can be distorted. Besides, it represents an additional expense of platinum wire and them the chambers are more expensive.
MiniTAFE equipments separate the S. cerevisiae chromosomes at high electric fields (22 V/cm), giving a suitable resolution between the bands of the electrophoresis patterns in the minigels (Riverón et al., Analytical Letters, vol. 28, p 1973-1991, 1995). Besides it, using the miniTAFE the S. cerevisiae chromosomes can be resolved in 5 hours, at 8 Volt/cm and 20° C. Small separation between opposite electrodes permits the construction of small chambers and the use of small buffer volume to cover the electrodes (350 ml). When across the miniTAFE electrodes a given voltage is applied, that is, a certain value of electric field strength ‘E’ is applied, then the heat dissipation is less than those obtained in TAFE equipments if the same ‘E’ would be applied. The samples plugs loaded into the minigels of the mini-equipments need small amount of biological material and the plug thickness ranges from 0.1 to 0.05 cm. They reduce the electrophoresis time and contribute to give sharp bands in the patterns (López-Cánovas et al., J Chromatography A, 806, p 187-197, 1998). In the minigels can be loaded as many sample plugs as permitted by the minigel width. For example, for a gel of 4.0×4.0×0.5 cm (width, height and depth) can be loaded up to 10 sample plugs of 2.5 mm in width and spaced apart 1 mm.
Despite of the mentioned advantages, the refereed equipments have inadequacies that limit their application in the analysis of numerous samples. In particular, when the number of samples to be analyzed changes considerably among the experiments. Several of these inadequacies are related to the shape and arrangements of the chamber dimensions as well as the existence of a single UEZ.
There are methods to select the run conditions in the PFGE equipments. For example, the CHEF Mapper from Bio-Rad has both auto-algorithm and interactive algorithm options (CHEF Mapper XA Pulsed Field Electrophoresis System. Instruction Manual and Application Guide. 31-40 Catalog Numbers 170-3670 to 170-3673. Bio-Rad). Both options permit to calculate the pulse time, the duration of the ramps of pulse time, reorientation angle, electric field and the optimum electrophoresis time to separate the DNA molecules of a given sample. In contrast to the auto-algorithm, that assumes constant values for the variables, the interactive algorithm permits to change the time, temperature and concentration of the buffer and the type and concentration of agarose. Both algorithms make the calculations based on empiric and theoretical data, collected during five years of experiences (Bio-Rad Catalogue. Life Science Research Products 1998/99. Bio-Rad Laboratories, 185). However, the manufacturers recommend entering to the auto-algorithm DNA sizes lower and larger than the limits of the range to be optimized. They also recommend to be considered that both algorithms can give erroneous results such as DNA mobility inversion in the mid-range of the gel, when extremely wide size ranges are entered in both algorithms.
There are other empirical expressions giving the pulse time that would separate a group of molecules which sizes are between a given size and other superior one called RSL (Resolution Size Limit) (Smith D. R. Methods I, 1990, 195-203). However, this relation is only valid in some experimental conditions and does not predict the resolution between any pair of molecules. There is also a function calculating the approximate conditions of the electric field and pulse time that separate a given set of molecules (Gunderson K. and Chu G. Mol. Cell. Biol., 1991, 11, 3348-3354). It should be noted, that such function only permits to estimate the approximate values of these two variables, but does not give the migration of the molecules at any experimental condition.
Despite many theoretical studies about the reorientation of the DNA molecules during PFGE have been performed (Noolandi J., Adv. Electrophoresis, 1992, 5, 1-57; Maule J., Mol. Biotech., 1998, 9, 107-126), they have not given practical results, useful in the laboratory. It means that they do not generated methods that permit the easy selection of the experimental conditions that separate a given group of molecules.
The equations proposed by López-Cánovas L. et al. (López-Cánovas L. et al., J. Chromatogr. A, 1998, 806, 123-139) to describe DNA migration in PFGE have not been extended to select the experimental conditions applicable in any equipment when the pulse time, electric field and temperature varies.
Up to now, the experimental conditions applicable in PFGE equipments are the result of the experience of the PFGE's user, more than the results of equations describing DNA migration in PFGE. There is not a secure method to predict the pulse and run times hat should be applied at any conditions. That method is particularly important when the minichambers of the mini-equipments are used, because in the miniequipments can be used high electric field strength. The use of such high electric field strength is not frequent in the rest of the PFGE systems.